JPH0335625B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides an improved digoxin immunoassay, and more particularly for a non-competitive heterogeneous immunoassay consisting of a labeled monovalent or divalent anti-digoxin antibody as an indicator reagent and an immobilized ouabain column as a means of performing the separation. Regarding the test kit. There is a large and widespread market for clinical laboratory diagnostic tests that can quickly and accurately measure the concentration of digoxin present in biological fluids. Digoxin is often present in nanomolar or sub-molar concentrations. In recent years, many immunoquantitative techniques have been developed for the measurement of clinically important ligands. Typically,
Competitive binding immunoassays consist of a conjugate of labeled substance bound to a binding component that participates in a binding reaction, producing two types of labeled conjugate: bound and free. The relative amounts of labeled conjugate, bound and free, are a function of the concentration of the ligand to be detected in the test sample. If the bound and free labeled conjugates are essentially indistinguishable by the means used to measure the labeled substance, the bound and free forms must not be physically separated. Must not be. This type of test is called a heterogeneous system. The two most widely used heterogeneous immunoassays are radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). In RIA, a sample containing an unknown amount of antigen is mixed with known amounts of radiolabeled antigen and antibody. The system is allowed to react to a point near equilibrium and then antibody-bound antigen is separated from unbound antigen. The sample antigen competes with the labeled antigen for a limited number of antibody binding sites, so the more antigen in the sample, the less labeled antigen will be in the bound fraction (or the unbound fraction). (increasingly). This method is generally time consuming (1-3 hours) and labor intensive. More recently, RIA has been automated by immobilization of antibodies on porous supports. After a sample suspected of containing antigen is mixed with a known amount of labeled antigen, the sample is passed through a column containing a limited number of immobilized antibody binding sites. Free or bound label can be quantified. Although rapid, this assay requires rigorous measurement of antibodies to make it reproducible. RIAs have two major disadvantages. The first used labeling substances are radioactive isotopes which have many problems regarding their handling, storage and disposal. Second, RIA is performed in a competitive manner (i.e., the analyte and labeled analyte compete for a limited number of binding sites on the antibody), and thus
The antibody affinity constant typically determines the sensitivity of the assay.
Limited to the range of 10 ^-8 M ¹ to 10 ^-11 M ¹ . ELISA works in principle except that the labeled substance is an enzyme rather than a radioisotope.
Similar to RIA. It is also limited by the fact that sensitivity is still a strict function of the antibody affinity constant. Other labeling substances have been described in addition to isotopes and enzymes. These include fluorophores,
Examples include coenzymes, bioluminescent substances, and enzyme inhibitors. Various methods of implementing the separation step in heterogeneous immunoassays are known. These include filtration, centrifugation, chromatography, and others. The use of affinity columns to carry out separation steps is described in French Patent Application No. 79 15992. It describes the use of a gel coupled thereto with a ligand that has an affinity for the labeled substance and also has molecular sieving properties. The use of gels that have affinity for the ligand of interest rather than the label and have molecular sieving properties has also been disclosed. The assays described can be performed in a competitive or non-competitive manner. U.S. Pat. No. 4,298,687 discloses a heterogeneous immunoassay method in which the substance to be measured is reacted with a labeled primary binding component, and the amount of unreacted binding component is determined to correspond to the primary binding component. It is measured by absorption onto a solid phase that provides specific binding for the component. This linear combination corresponding component is present in a limited amount. U.S. Pat. No. 3,654,090 discloses a non-competitive heterogeneous immunoassay for human chorionic gonadotropin (HCG), which uses an excess of enzyme-labeled divalent antibody and an immobilized antibody to carry out the separation step. transformation
An HCG column is used. This assay is limited in its sensitivity by the fact that it cannot distinguish between 1 molar HCG-bound antibodies and antibodies without HCG binding. Both species are retained by the affinity column. US Pat. No. 4,134,792 discloses a heterogeneous immunoassay method in which a label-specific binding counterpart for a ligand of interest is present in excess. The label-specific binding counterpart is a divalent antibody, which has the same disadvantages as described above. "British J. Haematol." Vol. 47, p. 269 (1981) discloses a two-site immunoradiometric assay (IRMA) for coagulation factors, in which monovalent Fab antibody fragments are used. The results show that a 10-fold higher sensitivity is obtained when using monovalent antibodies rather than divalent antibodies. U.S. Pat. No. 4,200,436 discloses an immunoassay method using labeled monovalent antibodies, in which an immobilized antigen (the same antigen that is to be measured) is used to separate the bound and free fractions. separate. Since it is primarily the bound fraction that is measured, this assay is usually performed in a competitive format. Sensitivity is therefore limited by the affinity constant of the antibody when the assay is performed in the preferred manner. In some cases, the analyte in the immunoassay can be replaced by an analyte congener. In general, performance is expected to be the same between the use of the analyte and the analyte congener. Surprisingly, it has been discovered that in the digoxin assay of the present invention, replacing digoxin with ouabain as the immobilized antigen significantly improves the sensitivity and accuracy of the assay. Although the exact mechanism leading to this improvement is not known, it is thought to be due to the nature of the antigen-antibody reaction. There is a need in the art for a heterogeneous immunoassay of digoxin whose sensitivity and accuracy are not limited by the affinity constants of the antibody. The non-competitive heterogeneous immunoassay method of the present invention consists of the following steps: (a) by contacting a molar excess of labeled monovalent or bivalent anti-digoxin antibody with a test sample suspected of containing digoxin; forming a reaction mixture such that one fraction of said antibody forms a complex with digoxin and one fraction remains free; (b) an amount of said reaction mixture sufficient to bind all of the free antibody; (c) separation of the free antibody from this reaction mixture by contacting the solid phase with ouabain immobilized on a solid support present in the complex eluted from the solid phase by measuring the label; including measuring the amount of The amount of digoxin in the sample can then be determined by comparing the standard curve. In other embodiments, competitive heterogeneous immunoassays are provided, which include the following steps:
(a) forming a reaction mixture by contacting a sample suspected of containing digoxin with a molar excess of ouabain immobilized on a solid phase; and (b) bringing this reaction mixture into contact with a molar excess of ouabain relative to digoxin. , except for ouabain, by contacting with a submolar amount of labeled monovalent or divalent anti-digoxin antibody, (c) allowing a reaction to occur whereby a fraction of the antibody forms a first complex with digoxin, and (d) separating the first fraction from the second fraction; and (e) either the first fraction or the second fraction. It involves measuring the amount of label present in one. It is generally desirable to immunologically purify antibodies before using them in immunoassays. animal serum,
Methods for isolation of IgG from ascites or tissue culture media and its immunological purification by affinity chromatography are also known in the art.
Briefly prepare the IgG fraction by ammonium sulfate precipitation. IgG fractions are then prepared by ion exchange, gel filtration or protein A chromatography. Affinity purification is performed by elution from an antigen column. Antibodies may be either polyclonal or monoclonal. Monovalent antibodies can be prepared by methods known in the art. for example
Fab fragments are obtained by papain digestion of IgG, and Fab' fragments are obtained by disulfide reduction of F(ab') ₂ fragments obtained by pepsin digestion of IgG. Any of a number of methods can be used for coupling a label to an antibody. The labeling substance is an enzyme,
It can be a radioisotope, chromophore, fluorophore, or any other substance that can generate a detectable signal alone or in combination with other reagents. Generally, at least one label should be coupled to each antibody, preferably covalently and in a manner that preserves the immunoreactivity of the antibody and the activity of the label. The free sulfhydryl group present in the Fab' fragment provides a specific reactive group for covalent attachment of the label. Heterobifunctional cross-linking reagents with maleimide or thiopyridyl groups are useful for this purpose. Generally, to ensure retention of immunological activity, it is desirable that the final step in the synthesis of labeled antibodies be an immunological purification step. Ouabain or its conjugate can be immobilized on a suitable carrier by methods known in the art, eg, according to Biohemistry, Vol. 9, p. 331 (1970). Generally, the carrier is chosen for its flow properties, and examples may include beaded agarose, beaded dextran, polyacrylamide or glass. Ouabain can be covalently attached to its carrier directly or through a spacer arm which can be a protein, a polyamino acid or a synthetic linking moiety. Typically, the affinity column material is discarded after one use, but it can be recycled if desired. It is generally undesirable for the carrier to have molecular sieving properties. That is, if the labeled antibody dissociates from the sample analyte,
This is because molecular sieves tend to reduce the likelihood that they will meet each other again. In a non-competitive format, the assay of the invention can be performed as follows. A known volume of patient sample, usually 5μ to 500μ of serum, containing an unknown amount of digoxin is mixed with a solution containing a labeled monovalent or divalent antibody in an apparent excess to digoxin. . Usually the labeled antibody is present in about 10-100 molar excess compared to digoxin. The digoxin and antibody are preincubated at a constant temperature between 4°C and 45°C, usually 37°C, for a specified length of time, usually at least 5 minutes, and less than 30 minutes. A known volume of this solution (usually 5 μ to 500 μ) containing digoxin-bound and unbound antibodies is passed through a column consisting of ouabain immobilized on a porous support, preferably having dimensions of 2 mm×10 mm. Enough ouabain-coupling carrier is used to bind all free labeled antibody. The column is eluted at a flow rate of 0.2 to 5.0 ml/min, usually with a total volume of 1 to 5 ml of an appropriate buffer.
The fraction eluted from the column contains labeled antibodies complexed with digoxin from the patient's serum. Label activity in this fraction is then measured. Alternatively, discard this fraction and add chaotropic agents or extreme PH values.
The retained antibodies can be eluted from the column by the chromatography. In the first case the amount of label is directly proportional to the digoxin concentration in the sample. In the second case it is inversely proportional. The assay method of the invention can be performed manually or it can be carried out using a variety of automatic or semi-automatic equipment, e.g.
Can be adapted to the ACA Independent Clinical Analyzer (manufactured by Dupont). In this case, the patient sample and excess labeled monovalent or divalent antibody are preincubated outside the device. Analyze a known volume of this mixture in a test pack (included here as a reference).
29725) automatically at the filling station of the device and then inject enough buffer to bring the final pack volume to 5 ml. The sample mixture is passed through a column of immobilized ouabain on a porous support placed in the pack header and eluted directly into the pack. This eluted fraction contains labeled antibodies complexed with the analyte in the patient's serum. This pack automatically adds the necessary reagents for the signal generation reaction in either a breaker/mixer or a breaker/mixer at 37°C and reads it out with a signal photometer. The assay of the invention can also be performed in a competitive format, meaning that the antibody is exposed to the samples digoxin and ouabain simultaneously rather than sequentially. In the competitive format, the assay is performed as follows. An aliquot of a sample suspected of containing digoxin, typically 10-100μ, is added to a test tube containing a molar excess of ouabain immobilized on a solid phase (generally an affinity column resin such as a cross-linked agarose). or dextran
100~1000μ filling capacity). A monovalent or divalent labeled anti-digoxin antibody solution is then added in a molar excess over the maximum amount of digoxin expected in the sample, but not more than a molar amount relative to ouabain. The amount of antibody solution is generally 5-50μ. The reaction mixture thus produced is incubated at a temperature of 23°C to 45°C, preferably 37°C, for 15 to 60 minutes, preferably 15 minutes, with gentle stirring. The antibody-digoxin complex is then separated from the antibody/immobilized ouabain complex. If affinity resins are used, centrifugation at 2000 xg for about 3 minutes is preferred. The supernatant fluid is then aspirated. Either the amount of label adsorbed onto the solid phase or the amount of label in the supernatant fluid can be measured to determine the amount of digoxin originally present in the test sample.
When the label is an enzyme, measurements can be performed by reacting the enzyme with its substrate to produce a detectable product. Non-enzymatic labels such as fluorophores, chromophores, and radioisotopes can be measured by techniques well known in the art. The advantage of using ouabain rather than digoxin on a solid phase is illustrated by the following example. In these examples, both digoxin (analyte) and ouabain (analyte congener) columns were prepared under various experimental conditions;
Their performance in immunoassays using digoxin affinity columns was then evaluated in terms of background, sensitivity and precision. Although the optimal conditions for the synthesis of each resin were different, the best ouabain resins consistently outperformed the best digoxin resins. Although only ouabain is specifically exemplified herein, equivalents are contemplated such as digitoxin, deslanoside, digoxigenin, and strophanthin. Example 1A Synthesis of Monovalent Antibody-Enzyme Conjugate Digoxin-specific antibodies were immunopurified directly from rabbit whole serum using ouabain-HSA immunoadsorbent. Ouabain was bound to the agarose matrix via a protein (HSA, human serum albumin) spacer arm. The first step involves the synthesis of the ouabain-albumin conjugate. Ouabain (0.56 mmol dissolved in 20 ml of water) was oxidized with sodium metaperiodate (1.02 mmol) for 1 hour at room temperature in the dark. Quantitative oxidation is ethyl acetate:methanol: _H2O (75:25:
This was demonstrated by thin layer chromatography on silica gel G plates developed in step 1).
3ml of Dowex AG-1X8 ion exchange resin
Excess periodate was removed by passing the aqueous mixture over a column. Quantitative recovery of ouabain was demonstrated by tracking radiolabeled (tritiated) ouabain. 0.4
Buffer the solution of ouabain oxide to pH 9.5 by adding ml of 5% Na ₂ CO ₃ ,
It was then combined with 20 ml of HSA solution (28 mg/ml).
After 45 minutes, the conjugate was reduced by adding 0.3 g of sodium borohydride freshly dissolved in 20 ml of water. After 3 hours, 8 ml of 1M formic acid was added to lower the pH to 6.5. After leaving it at PH6.5 for 1 hour, 1 mol,
The PH was raised to 7.5 with _NH4OH . The entire reaction mixture was thoroughly dialyzed against distilled water and finally
0.015M Sodium Phosphate Buffer (PH7.8),
Dialyzed against 0.15M NaCl. This conjugate was concentrated using Amicon PM-30 membrane to yield 4.2mg/
ml. Protein concentration was determined by the Lowry method known in the art. Ouabain-HSA conduit gate in "Bio-"
Afuigel 10 (manufactured by Bio-Rad Laboratories) using the method described in the Radmanual.
I made it immobile above. 25ml Afui Gel 10 to 75
ml of ice-cold water. This gel was added to the dialyzed ouabain-HSA conjugate and mixed overnight on a rocker at 4°C. Excess active ester groups were quenched by adding 0.1 ml of 1 molar ethanolamine (PH 8.0) for 1 hour at room temperature. Finally, completely remove this gel with distilled water.
Then in order: 500 ml of 0.5 molar NaCl, 400 ml of 0.1 molar glycine (PH2.5), 300 ml of 2.5 molar
_NH4SCN , washed with 1000ml phosphate buffered brine. 6ml of ouabain hydrophilic resin
Column (0.7 x 15cm) so that the bed volume is
and equilibrated with phosphate buffered saline. Antiserum (10 ml of Capel anti-digoxin serum at 4.5 mg/ml monospecific antibody) was applied at a flow rate of less than 1 ml/min. Column at 280nm
with phosphate buffered saline until the absorbance reached baseline (<0.01). Then 60ml
The antibody was eluted from the column with 3 molar NH ₄ SCN (PH 7.5) and immediately dialyzed four times against diphosphate buffered saline at 4°C. The 27 ml of affinity purified anti-digoxin antibody was concentrated to 2.7 ml on an Amicon stirred cell apparatus (PM-30 membrane). Final protein concentration is 10mg/
It was hot in ml. The sample was dialyzed against 1000 ml of 0.1 molar sodium acetate (PH4.5) for 4 hours at 4°C. After dialysis, 20 μ of a 10 mg/ml pepsin solution dissolved in the same sodium acetate buffer was added and the temperature was raised to 37° C. and maintained for 20 hours. After this digestion time, the sample was clarified by brief centrifugation and then equilibrated in 0.015 molar sodium phosphate (PH 7.4), 0.15 molar NaCl (phosphate buffered saline).
Chromatography was performed on a −150 column (1.5×90 cm). Column fractions containing the (Fab′) ₂ fragment identified by gel electrophoresis were pooled (19.2 ml) and then subjected to pressure filtration (PM-30
Amicon membrane) to concentrate to 2.7 ml. After concentration, this (Fab') ₂ fragment was reduced to the corresponding Fab' fragment by adding 55μ of 1M dithiothreitol solution. The reduction is
It was carried out for 90 minutes at 25°C. Then at 4°C 0.15M NaCl, 0.015M Sodium Phosphate, pH 5.6 (2
Dialysis was performed with 1000 ml). The Fab' fragment thus prepared was then reacted with a 20-fold molar excess of m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS). 2 ml of 85μ of a 79 mmol MBS solution in tetrahydrofuran
Add to the solution of Fab′ fragments and at 25 °C.
The reaction was carried out for 1 hour under argon. This mixture was added to a cephadex G in phosphate buffered saline.
Column (1.5 x 40
cm). The derivatized Fab′ fragments eluted into the void volume were pooled and 12 min in phosphate-buffered saline at 4°C.
Combined with 2 ml of mg/ml β-galactosidase.
After 16 hours the solution was concentrated to 2 ml in an Amicon PM-30 pressurized stirred cell and then loaded with Sepharose 4B-CL (bead-shaped cross-linked macroparticles with a 1-5 x ¹⁰⁶ Dalton exclusion limit in a 1.5 x 90 cm column). Porous agarose) column chromatography was performed. The Fab'-β-galactosidase conjugate was co-eluted with free β-galactosidase. All peaks of enzyme activity were pooled and then immunologically purified on an Ouabain affinity column. The immunological purification method was as follows. The pooled column fractions from the Sepharose 4B-CL column were eluted through an Ouabain Affinity column (1.0 x 7.0 cm) and then with 100 ml of phosphate buffered saline. The Fab'-β-galactosidase conjugate was then eluted with 50 ml of 23 mmol ouabain in phosphate-buffered saline. This eluate corresponds to the final reagent, which was
Dialyzed six times against phosphate buffered saline at 4°C. B. Synthesis of Ouabain and Digoxin Resin Ouabain and digoxin were separately synthesized in various ratios via the organic spacer component triethylenetetraamine (TETA) to Sephadex G-10 (a bead-type dextran available from Pharmacia Fine Chemicals). I made it couple. Ouabain (20-325 mg) was dissolved in distilled water and oxidized for 2 hours at 25°C by adding a 5-fold molar excess of sodium metaperiodate. At the end of this time the reactions were stopped by passing each solution through a 5 ml column of Dowex I-X8 (Cl ^- ion exchange resin) which removed excess periodate. The eluate was collected and brought to a final concentration of 0.1 molar sodium phosphate (PH 7.0) by addition of concentrated stock solution.
TETA (50-125 mg) was added to each solution (see Table 1A) and the PH of the final solution was adjusted to PH 7.0.
This solution was incubated for 1 hour at 25°C and at the end of this time 30mg of sodium cyanoborohydride was added. The resulting solution was stirred at 25°C for 48 hours. The same method described above for ouabain was followed by
However, the test was performed on digoxin by dissolving it in 30% ethanol. Details of the experiment are shown in the table.
Shown in 1B. Each conjugate (9 lots of ouabain-TETA and 9 lots of digoxin-TETA) was then coupled to Sephadex G-10 by the following method. 210 g of Cephadex G-10 was swollen in 1-distilled water. The resin was then washed three times with one portion of water. Finally, the resin was oxidized by suspending it in 1 sodium metaperiodate (10 g/). 25℃ for 1 hour
After being allowed to settle, the resin was allowed to settle, the supernatant fluid was removed, and the resin was washed on a sintered glass funnel using approximately 3 portions of 0.25 molar sodium phosphate (PH 7.0). The resulting resin was divided into 25 ml portions and one portion was mixed into each lot of the synthetic conjugate. After mixing for 1 hour, sodium cyanoborohydride (30 mg) was added to each portion of the conjugate-loaded resin. The resulting suspension is mixed at 25°C for 64 hours, during which time the resin is allowed to settle, the supernatant fluid is removed, and the resin (each section separately) is mixed with 300 ml of water, 300 ml of 0.5 mol.
Washed with NaCl and 500 ml 0.15 molar sodium phosphate (PH 7.1). Each resin section was loaded into an ACA Independent Clinical Analyzer analytical column (0.5 x 8 cm, 1.8 ml per column). The column was then loaded with aca containing 7 mg of o-nitrophenyl-β-D galactopyranoside (ONPG).
A dimple 6 was placed in the water tube of the Independent Clinical Analyzer Analysis Test Pack. C. Comparison of ouabain-TETA-cephadex and digoxin-TETA-cephadex Each resin lot synthesized in (B) above was evaluated on the ACA Independent Clinical Analyzer according to the following protocol. 10 pmol of β-galactosidase-labeled anti-digoxin synthesized in (A) above.
Fab' was added to 100 μ of human pooled serum samples containing 0 or 5 ng/ml digoxin. After incubation for 10 minutes at 25°C, the antibody-sample mixture was automatically injected into the ACA Independent Clinical Analyzer analytical test pack and eluted through the column in the pack water tube. Following the sample, add 2 ml of
0.15M sodium phosphate (PH 7.8) was flushed.
Column flow rate was 34μ/sec. This pack was then filled with an additional 2.9 ml of water at needle position 2 (column bypass). About 3 1/2 ONPG
After a few minutes, the breaker/mixer was discharged. Enzyme activity was measured at 405 nm at 29 and 46 seconds after substrate addition. The table compares the performance of all 18 resin lots in terms of background (Table A) and sensitivity (Table B). The background is when the sample contains 0ng/ml digoxin.
It was defined as the change in absorption at 405 nm. Under these conditions all labeled antibody should remain bound to the column. Sensitivity as separation, i.e.
It is defined as the change in absorbance at 405 nm between 0 ng/ml digoxin and 5 ng/ml digoxin. Although the background was generally lower when the column consisted of digoxin-TETA-cephadex, the sensitivity was always better when the column consisted of ouabain-TETA-cephadex. The best ouabain resin had a sensitivity of 0.158 absorption units/min, while the best digoxin resin exhibited only 0.110 absorption units/min. Those resins marked with an asterisk in Table B were selected for further study. D. Accuracy The resins marked with an asterisk in the table, one consisting of ouabain-TETA-cephadex and the other consisting of digoxin-TETA-cephadex, were further evaluated for assay accuracy against digoxin. 0, 1
Alternatively, packs were run exactly as described above using samples containing 3.4 ng/ml digoxin. At least 13 packs were run at each drug concentration. The mean value (X), standard deviation (SD) and coefficient of variation % (CV) were then determined. These are shown in the table. 1n, which is the medically determined concentration for this drug
The precision of the measurement in g/ml digoxin was significantly improved for the ouabain column compared to the digoxin column. When using a digoxin column, a person with a serum concentration of 1 ng/ml digoxin is easily misidentified. and possibly lead to over-dosing (or under-dosing) of the drug. The consequences of such measurement errors can be significant, as digoxin is ineffective at concentrations below 1 ng/ml and toxic at concentrations significantly above that. For this reason, the use of an ouabain column instead of a digoxin column forms the basis of the invention.

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【table】

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[Table] Example 2 A Synthesis of bivalent (F(ab') ₂ ) antibody-enzyme conjugate 1 ml of affinity purified anti-digoxin F
(ab') ₂ fragments (prepared as described in Example 1) [2.85 mg/ml protein - 0.015 M sodium phosphate, 0.15 M NaCl, 1 mmol EDTA
(in pH 7.0)] at 23-25°C with 9.1μ of 60 mmol succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate (SMCC) solution dissolved in dimethylformamide. I let it happen. After 60 minutes, add a Sephadex G-25 column (1.5 x 30
The reaction was stopped by desalting the solution over cm). The eluted protein in the void volume was collected and concentrated to 1 ml using an Amicon stirred cell concentrator (PM-30 membrane).
1 ml 0.05 M Tris HCl, 0.15 M NaCl, 1
24 mg of β-galactosidase dissolved in mmolar MgCl ₂ (PH 7.5) was added to the F(ab') ₂ -SMCC adduct and reacted for 20 hours at 4°C.
The reaction was quenched by the addition of 10μ of 0.1M 2-mercaptoethanol for 1 hour at 4°C. F(ab') ₂ -β-galactosidase conjugate in 0.05 mol Tris HCl, 0.15 mol
Sepharose 4B _column (1.5 x 90
This F by chromatography on cm)
The (ab′) ₂ -β-galactosidase conjugate was separated from unreacted β-galactosidase. B. Synthesis of Ouabain Resin and Digoxin Resin Ouabain and digoxin were each coupled separately to Cephadex G-10 at an optimal ratio via bovine serum albumin (BSA). (1) Ouabain-BSA is 500ml of hot distilled water (70ml
℃) by dissolving 5 g of ouabain octahydrate. After the solution was cooled to 25°C, 7.3g of sodium metaperiodate was added and then mixed continuously for 2 hours at 25°C in the dark. The oxidation was then stopped by passing the mixture through a 250 ml bed of Dowex (1-X8) anion exchange resin. The eluate was collected and combined with a solution of bovine serum albumin (10 g/500 ml) dissolved in 1 molar sodium phosphate buffer (PH 7.0). 1 at 25℃
After an hour, 0.64 g of sodium cyanoborohydride was added with stirring and the mixture was incubated at 25° C. for 72 hours. The ouabain that did not couple with the ouabain.
The BSA conjugate solution was removed from the mixture by dialysis for 24 hours against running distilled water and then against 20 volumes of 0.015 molar sodium phosphate buffer (PH 7.0) at 4°C. Before coupling to Sefdex resin
The final ionic strength of the conjugate solution was adjusted to 0.25 molar by adding 14.6 g of NaCl. (2) Ouabain - BSA's Safety Index G -
Coupling to 10 Sephadex G-10 (420 g) (manufactured by Pharmacia Fine Chemicals) was swollen in 2000 ml of distilled water for over 1 hour. The resin particulates were removed by decanting and resuspending in 3 x 2000 ml of water. Then add 20 resin
The mixture was oxidized by resuspension in 1000 ml of water containing g of dissolved sodium metaperiodate. After 10 minutes, the resin was washed with 5 x 2000 ml of water, then
Washed with 4000 ml of 0.25 molar sodium phosphate buffer (PH 7.0). Decanted resin
It was resuspended in 1000 ml of ouabain-BSA solution (as above) and mixed for 1 hour at 25°C, then mixed with 0.66 g of sodium cyanoborohydride. After 72 hours, underwater
0.1% Sodium Dodecyl Sulfate 4000
12 ml of distilled water and then thoroughly washed with 4000 ml of 0.15 molar sodium phosphate buffer (PH 7.1). The final resin was loaded as a slurry into small columns (0.5 cm x 7 cm) for use in automated analysis in a Dupont ACA Independent Clinical Analyzer. (3) Digoxin-BSA in 75ml of ethanol
It was prepared by dissolving 1.25 g of digoxin and then combining this solution with 150 ml of water containing 1.83 g of sodium metaperiodate. After stirring for 2 hours at 25°C, the oxidation was stopped by passing the mixture through a bed of Dowex (1-X8) anion exchange resin (100ml). The eluate was combined with a solution of bovine serum albumin (2.5 g) dissolved in 0.1 molar sodium phosphate (PH 8.5). Sodium cyanoborohydride (0.24 g) was then added;
This mixture was then allowed to mix at 25°C for 48 hours. Free, unconjugated digoxin was dissolved in running distilled water for 2 days, then 20 vol.
0.015M Sodium Phosphate Buffer (PH
7.0) at 4°C. (4) Digoxin-BSA Cephadex G-
Coupling to 10 Sephadex G-10 (50 g) was swollen in 250 ml of distilled water for over 1 hour. The resin particulates were removed by decanting and resuspending. The resin was then oxidized by resuspending it in 250 ml of water containing 5 g of dissolved sodium metaperiodate. After 10 minutes, add 5x resin.
Washed with 250 ml of water followed by 1000 ml of 0.1 molar sodium phosphate buffer (PH 8.5). The decanted resin (~250 ml settling bed volume) was then slurried with 125 ml of 0.1 molar sodium phosphate buffer (PH 8.5) containing 125 mg of sodium cyanoborohydride.
After 24 hours of constant mixing, the resin was mixed with 3 x 500 ml of water, 500 ml of 0.15 molar sodium phosphate (PH
7.8) and resuspended in 125 ml of 0.15 molar sodium phosphate (PH 8.5) at 4°C.
Add anhydrous lower acid (1.25 ml) to the slurry and
The reaction was carried out at 4° C. with mixing for 30 minutes. The resin was mixed with 1 part 0.5M NaCl, 4 parts distilled water and 2 parts
Wash thoroughly with 0.15M sodium phosphate (PH7.1). A small column (0.5 cm
× 8 cm) was filled in slurry form. C. Comparison of Digoxin-BSA-Sephadex Resin and Ouabain-BSA-Sephadex Resin Using Bivalent Antibody Enzyme Conjugate Both resin lots were compared under the same conditions. Said
(A) Synthesized F(ab') ₂ -β-galactosidase (2.6 pmol in 200μ buffer) in normal subjects containing various amounts of digoxin (0, 0.5, 1.5, 3.5 and 5.0 ng/ml) serum standard solution
Added to 200μ. After an incubation period of 10 minutes at 25°C, the total resistance-sample mixture is automatically aca
It was injected into an Independent Clinical Analyzer test pack and eluted through the column in the pack water tube. The sample was followed by 2 ml of 0.15 molar sodium phosphate pH 7.8. Column flow rate is 34μ
/ seconds. The pack was then filled with an additional 2.6 ml of water at needle position 2 (column bypass). ONPG was released from the breaker/mixer after approximately 3.5 minutes. Enzyme activity is 405nm;
Measured at 29 and 46 seconds after substrate addition. FIG. 1 and the table compare the performance of two optimized digoxin-BSA-Sephadex and ouabain-BSA-Sephadex resins in terms of background and slope sensitivity (FIG. 1) and precision (Table). Although both resin lots allow a linear dose response for the F(ab') ₂ -β-galactosidase conjugate, the ouabain resin has a lower background (32% lower) and a greater slope sensitivity (47%). large). Lower background and higher slope sensitivity are preferred for better assay performance and precision. Moreover, the assay accuracy was significantly better for ouabain resin than for digoxin resin (Table). Individual pack assays were performed exactly as before using two digoxin concentrations, 0.5 ng/ml and 0.5 ng/ml.
Performed at 1.5ng/ml. At least 12 packs were run for each drug concentration. and mean value (X), standard deviation (SD) and coefficient of variation (%)
(CV) was measured.

【table】 [Brief explanation of drawings]

The attached drawing is a graph showing the slope sensitivity when using a digoxin column and an ouabain column.

Claims (1)

[Claims] 1. A test kit for heterogeneous digoxin immunoassay for measuring the amount of digoxin in a liquid sample, comprising a labeled monovalent or divalent anti-digoxin antibody and a solid phase having immobilized ouabain.